Arctigenin, a lignan extract from Arctium lappa (L.), exhibits anti-inflammation, antioxidation, vasodilator effects, etc. However, the effects of arctigenin on bronchus relaxation are not well investigated. This study aimed to investigate how arctigenin regulates bronchus tone and calcium ion (Ca2+) flow. Trachea strips of guinea pigs were prepared for testing the relaxation effect of arctigenin to acetylcholine, histamine, KCl, and CaCl2, respectively. Furthermore, l-type calcium channel currents were detected by patch–clamp, and intracellular Ca2+ concentration was detected by confocal microscopy. The results showed that arctigenin exhibited relaxation effect on tracheae to different constrictors, and this was related to decreasing cytoplasmic Ca2+ concentration by inhibiting Ca2+ influx partly through l-type calcium channel as well as promoting Ca2+ efflux. In summary, this study provides new insight into the mechanisms by which arctigenin exhibits relaxation effect on bronchus and suggests its potential use for airway disease therapy.